Holographic sensors and their production

ABSTRACT

A method for the production of a holographic sensor wherein the holographic recording material forming the sensitive element is a polymer matrix, which comprises diffusing into the matrix one or more soluble salts that undergo reaction in situ to form an insoluble sensitive precipitate; and recording a holographic image. This method allows the production of a holographic sensor wherein the holographic recording material forming the sensitive element is an insoluble polymer film.

FIELD OF THE INVENTION

This invention relates to a chemical sensor based on a sensitive elementwhich is a hologram made from a silver halide-based recording material.

BACKGROUND TO THE INVENTION

Many different approaches to producing chemical sensors have beendescribed in the academic and patent literature. There exists a widerange of different transduction mechanisms, which can be married with asuitable chemical or biochemical interface to realise a more or lessselective sensor capable of identifying and/or quantitating a particularchemical substance. Transducers that have been successfully appliedinclude those harnessing electrical, acoustic or optical phenomena.

WO-A-9526499 discloses a new class of optical sensor, the holographicchemical sensor, based on a volume hologram. This configuration has theunique feature that the analyte-sensitive matrix film has the opticaltransducing structure disposed throughout its volume. Because of thisphysical arrangement of the transducer, the optical signal generated bythe sensor is very sensitive to volume changes or structuralrearrangements taking place in the analyte sensitive matrix as a resultof interaction or reaction with the analyte.

In order to realise a holographic chemical sensor, a hologram must berecorded in a material which responds to a certain chemical orbiochemical analyte. To date, however, few suitable holographic filmmaterials exist. The most common type of holographic film is a silverhalide-containing gelatin film made by a liquid phase colloid formationtechnique, followed by coating onto a suitable support layer. Thismethod has been universally adopted for production of ultra-fine grainsilver halide film and works well with gelatin as the support polymer.To be successful, this method requires that the polymer that will formthe support matrix should be:

soluble in a solvent compatible with silver and halide salts (usuallywater);

a stabiliser of growing silver halide colloidal particles;

capable of forming a film when cast on a support substrate that isstable; and

insoluble under the conditions required to process the film.

Millington et al, Anal. Chem. 67:4229-33 (1995), Millington et al,Sensors and Actuators B33:1-5 (1996), and Blyth et al, Anal. Chem.68:1089-94 (1996), describe the applications of gelatin-basedholographic film to detect water in hydrophobic liquids and proteolyticenzymes such as trypsin.

Another known type of hologram recording material is dichromated gelatinor dichromated polyvinyl alcohol (PVA). These materials contain nosilver halide and the holographic image is recorded by a photo-chemicalcross-linking process which renders the material harder and less able tobe swollen by solvents in regions exposed to light. Subsequent chemicalprocessing produces small air voids in the material, creating amodulation of the refractive index and producing very bright holograms.

A third group of holographic recording materials is the photopolymermaterials; see Mannivanen and Lessard, Trends Pol. Sci. 2:282-90 (1994).These vary widely in their composition and mechanisms of hologramrecording. The materials from which they are made and their structuresrender them unsuitable for sensing applications.

In the early days of photography, before the liquid-phase colloid methodwas introduced, an alternative method for making photosensitive film wasintroduced by Henry Fox Talbot; see GB-A-0012906 (1849) and GB-A-0013664(1851). This was subsequently optimised for gelatin film, as describedby Liesegang, Phot. Rund. 52:198-200 (1915). In this procedure, aprotein film was first made and then treated with silver salt, dried andtreated with a halide salt or molecular halogen. The order of exposureto silver ions and halogen could also be reversed.

Very few polymers other than gelatin have the property of being able tostabilise silver halide colloids in solution. Hence gelatin is stilluniversally used in the making of fine-grain silver halide film, despitemany attempts within the photographic industry to find viablealternatives, as described by Croome, J. Photo. Sci. 30:181-5 (1982).

Applications of gelatin to chemical sensing are severely restricted byits complex chemical nature and hence the difficulty of chemicallyderivatising it in a rational way in order to change its analyteselectivity. PVA-based holograms have some advantages over gelatin, butthey are still very restricted in their scope, due to theincompatibility between the sensitive silver halide colloidal particlesand the conditions required to chemically transform the PVA into astable and analyte-selective matrix.

Other types of holographic recording material known to those skilled inthe art are also unsuitable for a variety of reasons. For example, whenholograms made by the dichromating method are immersed in liquid, thevoids fill up and the refractive index modulation is lost; hence thesematerials are entirely unsuitable for use as liquid phase chemical orbiochemical sensors. Photopolymer holograms are either uncross-linked orare only cross-linked in the areas of light exposure during hologramrecording, and thus are not stable to immersion in solvents of thepolymers from which they are made. Such solvent exposure disrupts theordered layer structure of the hologram. Many photopolymer formulationsare also very hydrophobic and hence incompatible with aqueous solutionsof analytes, which are incapable of penetrating the hologram.

Due to the problems with other types of holographic recording material,and also because of ease of handling and unrivalled sensitivity tolight, silver halide-based films remain the materials of choice forrealising holographic chemical sensor devices. However, the liquid phasecolloid method is inapplicable to the types of custom-designed polymermaterials which are required for analyte-selective chemical sensing.This is because the chemical conditions required to make suitablecustom-designed polymer films are not compatible with the silver halidecolloid formation process, and often lead to insoluble cross-linkedmaterials.

SUMMARY OF THE INVENTION

The present invention addresses the need for an alternative method ofproduction of silver halide-based holographic recording material. Thisis achieved by using a sequential treatment technique similar to thatdescribed by Talbot, where the polymer film is made first, and thesensitive silver halide particles are added subsequently. This approachis combined with materials that have not previously before used forholographic recording.

According to a first aspect of the present invention, a method forpreparing a holographic sensor of the type wherein the holographicrecording material forming the analyte-sensitive element is a non-rigidpolymer matrix, comprises diffusing soluble salts into the matrix wherethey react to form an insoluble light-sensitive precipitate with aparticle size less than the wavelength of light; a holographic image maythen be recorded.

This method can be used to produce volume holograms, of the general typedisclosed in WO-A-9526499, suitable for use as sensors. The polymermatrix may be gelatin, but an advantage over the prior art is thatother, better defined polymers can also be used. For example, the matrixmay be an insoluble polymer film. The matrix may have any of thefollowing advantageous characteristics (many of which are distinct fromthose of gelatin):

a defined pore volume specific for the analyte or a component thereof;

hydrophobicity;

homogeneity;

inertness with respect to any material reactive with gelatin;

non-charged;

requires processing, during or after formation, that is incompatiblewith the presence of the photosensitive substance;

cannot stabilise silver halide colloids in solution;

has a structure comprising essentially only regular repeating units.

According to a further aspect of the invention, a sensor for an analytecomprises a hologram supported on or within an insoluble polymer film,wherein at least one optical characteristic of the hologram varies as aresult of variation of a physical property occurring throughout the bulkof the matrix.

DETAILED DESCRIPTION OF THE INVENTION

The invention preferably uses as its support a transparent glass orplastics substrate which has been pre-treated or “subbed” to improve theadhesion of the overlying polymer layer which will support theholographic structure. The nature of the pre-treatment depends on thesubstrate material and the polymer that will overlay it. Many techniquesare known to those skilled in the art, including silanisation of glassand U.V., thermal or chemical bonding of thin polymer layers to thesurface. The most appropriate method is chosen for the combination ofmaterials being used.

A film of the polymer of interest for a particular sensing applicationis then deposited on top of the pre-treated substrate. The polymer istypically any that has a regular structure of the type comprising, say,at least 50 or 100 repeating units of the same basic structure in theprimary chain, whether a homopolymer or copolymer. Typical polymers foruse in this invention are selected from polyvinyl alcohol,polyvinylpyrrolidone, polyhydroxyethyl acrylate, polyhydroxyethylmethacrylate, polyacrylamides, polylmethacrylamides, homopolymers orcopolymers of polymerisable derivatives of crown ethers, and esters ofor co- or terpolymers of polyhydroxyethyl acrylate, polyhydroxyethylmethacrylate, polymethacrylamide or polyacrylamide, optionally withother polymerizable monomers or cross-linkers.

The polymer may be soluble or insoluble. If soluble, it may be depositedas a film by any of the techniques known for this purpose, such asspin-coating, roller-coating or use of metering rods or doctor blades.The polymer solution used for coating may have chemical cross-linkingagents included, in order to render the resulting film tough andinsoluble after curing, or the dried film may subsequently be immersedin a bath of a cross-linking agent to render it insoluble. A typicalexample of this procedure comprises coating an aqueous polyvinyl alcohol(PVA) solution containing a small amount of glutaraldehyde and a traceof acid catalyst to make a stable cross-linked PVA film.

In an alternative approach to making the polymer film, a mixture ofappropriate polymerisable monomers can be mixed with thermal, U.V. orvisible light initiators and optionally with solvents, and polymerisedin situ on top of the substrate. Typical examples of this approachutilise co- or ter-polymers of acrylate, methacrylate oracrylamide-based monomers, preferably mixed with a certain amount ofcross-linker to give a stable insoluble film capable of supporting aholographic structure within it.

Regardless of the film formation method, and after appropriate curingprocedures, a stable polymer film can be obtained, that adheres to thesubstrate. The dry thickness of the film is typically 5 to 50 μm,although it could be thicker or thinner if appropriate.

After film formation, the film is optionally washed with a suitablesolvent to remove any soluble residues, and may be subjected to furtherchemical derivatisation steps if appropriate, before proceeding with theincorporation of the photosensitive silver halide particles andconstruction of the holographic structure within the film.

The polymer film is preferably soaked in a solution of a silver salt.This is typically at a concentration of 0.1 to 0.5M. The solvent useddepends on the hydrophobicity of the polymer film. For hydrophilicfilms, aqueous silver nitrate can be used, but, for more hydrophobicmaterials, solutions of organic soluble silver salts such as silverperchlorate in solvents such as propan-1-ol give much more efficientpenetration into the film and hence better silver density in theresulting holograms. The soaking time depends on the nature of thepolymer film and can range from less than a minute to hours.

After soaking in a silver salt, the film is optionally dried. The filmis then dipped in a bath containing a halide ion. This and subsequentsteps must be carried out under safe lighting. By choice, the halidesalt is sodium bromide, but chloride or iodide or a mixture, or lithiumor potassium salts, may also be used. A sensitising dye matched to thewavelength of the laser that will be used to record the holograms may bealso included in the, say, bromide bath. This can be omitted and thehologram sensitised by a post-treatment in a dye solution, but thephoto-sensitivity is generally better if the dye is included with thebromide. The bromide bath is preferably agitated, in order to minimisesurface build-up of precipitated silver halide.

Immersion time in the bromide bath is very dependent on material. Forsome polymers, such as polyacrylamide, the time may be very short, e.g.15-30 seconds. For most materials, a few minutes is optimal, but somematerials require longer. The bromide bath can also optionally containmethanol or another water-miscible solvent. In this case, it willusually be necessary to substitute LiBr for NaBr for solubility reasons.The solvent aids penetration of bromide ions into some types of polymerfilms such as those made from poly(HEMA).

The order of adding the silver salt and the halide salt to the polymerfilm can be reversed without significant changes to the results. Theorder described is preferred because it minimises the amount ofexpensive silver salts involved.

After removal from the bromide bath, the film is washed in water toremove soluble ions and is then exposed to laser light in an appropriateoptical configuration. The film can be exposed wet or dry or in anypartially swollen state, depending on the final application and thedesired reflection colour of the hologram. The degree of swelling duringexposure can be used to tune the colour. The holographic exposure can bemade using any of the configurations known to those skilled in the art,but a preferred format is a simple reflection hologram made using aplane mirror as the object.

Following exposure, the hologram is developed using an appropriatedeveloper. This can be selected from the wide range of formulations usedin holography. For some materials (particularly more hydrophobic ones),a developer containing methanol gives superior results. Afterdevelopment, the film is washed thoroughly with water.

In most cases, it is desirable to fix the developed hologram to removeresidual silver halide. This is most conveniently achieved using sodiumthiosulphate solution, with the optional addition of alcohol for morehydrophobic materials. Fixing typically requires about 5 minutes butdepends on the nature and thickness of the polymer film.

Finally, the hologram may optionally be bleached. Bleaching makes thehologram near-transparent and is helpful if the transmitted spectrum ismeasured rather than the reflected spectrum. Appropriate bleachingconditions can be chosen from the range of options familiar to thoseskilled in holography. The preferred configuration for the novelholographic sensors is to use them in reflection measurements. In thiscase, it is preferable to leave the holograms unbleached since they aremore light-stable in this state.

The completed hologram can be used in any appropriate monitoring format.This could be a transmission or reflection spectrometer device, adip-stick, a fibre-optic probe or a label. These are given by way ofexample only.

The design of the polymer material from which the hologram isconstructed is the key to the analyte-selective sensing abilities of thefinal device. Many different design approaches could be applieddepending on the target analyte and a few will be described here by wayof example, although the scope of the invention should be understood tobe very broad and is not limited only to the approaches described below.

One approach is to make the hologram in a natural or synthetic polymer,or a mixture containing one or more such polymer(s), which can bedegraded by an enzyme or a group of related enzymes. When the enzymehydrolyses the polymer, the structural integrity of the polymer film isundermined, and the reflection spectrum of the hologram changes giving asignal. Example 1 (below) describes this approach, using starch toselect for an enzyme, α-amylase, which specifically degrades starchchains. By replacing the starch with other carbohydrates, theselectivity would be altered. Thus dextran holograms would select fordextranase and pullulan holograms would select for pullunases andiso-amylases. By using other types of polymers, other classes of enzymesmay be targeted, such as proteases.

It is not necessary for the whole of the polymer structure to bedegradable by the target enzyme. Only occasional linkages along the mainchain of the polymer need to be cleavable, or alternatively,cross-linking sites can be targeted. Example 5 (below) shows howcleavage of the cross-links in a gel structure leads to a signal. Thissimple chemical example can be extended by designing more complexcross-links, such as ones with peptide spacers containing cleavage sitesfor specific proteases. These designed synthetic polymer films may thushave specificity for particular proteases.

By coupling short peptide protease substrates to the polymer chains ofthe film, it may also be possible to create a response by a chargechange mechanism. For instance, if the peptide was initially unchargedbut created an immobilised charged group when cleaved by the protease,the increase in immobilised charge groups may cause the film to swell,hence generating a response. The converse situation, where a chargedgroup is removed by enzymic cleavage, could also be exploited. In thiscase, a contraction would be observed. This concept can also be extendedto other classes of enzymes using appropriately designed substrates.

The design concepts for holographic sensors are not limited to systemsusing cleavage mechanisms. If the analyte of interest changes themicroenvironment around a particular type of polymer chain, it may causethe polymer chain to change its conformation, leading to a measurablevolume change. This is demonstrated by Example 2 (below), where additionof ethanol to a poly(HEMA)-based hologram causes a progressive swellingwhich can be used to quantitate the amount of ethanol present.

By inclusion of a specific molecular receptor in the polymer structure,volume changes can be induced upon binding of the molecule in question.This is demonstrated in Example 3 (below) where crown ethers are used asreceptors that can specifically bind certain types of metal ions. Theresponse can arise by various mechanisms. Osmotic swelling may occur ifcharged groups are held in the gel by the receptor. Alternatively, ifbinding of the analyte causes dimerisation of the receptor, this willtend to pull the receptors closer together and cause a contraction ofthe film. Binding of the analyte in the molecular receptor may alsocause twisting of the polymer chains or otherwise alter the localmicroenvironment within the polymer film such that a measurable volumechange results. Materials such as crown ethers may provide a definedpore volume within which specific analytes are received.

More complex polymer materials may also be designed, where the structureis, at least in part, held together by an interaction between animmobilised analyte and a molecular receptor for that analyte. Thisgenerates physical cross-links in the polymer structure. Uponintroduction of free soluble analyte, this will compete with theimmobilised analyte for the binding sites in the molecular receptor,leading to a reduction in the physical cross-linking of the film andconsequently to swelling. This approach may be used with a range ofmaterials and analytes, particularly for antigen/antibody interactions,ligand/binding protein interactions (e.g. glucose with concanavalin A),and interactions of designed artificial receptors with analytes.

For realisation of the many designed materials that could be created formeasuring particular analytes, the in situ polymerisation approachdescribed above is preferred, because of the ability to control anddesign the properties of the resulting polymer film. Specially designedimmobilised substrates, receptor molecules, cross-linkers etc. caneasily be synthesised and incorporated into films by copolymerisationwith other suitable monomers. The degree of swelling can be controlledby alteration of the concentrations of monomers or cross-linkers in thesystem; hence the behaviour of a hologram can be fine-tuned to match theapplication, for example with respect to sensitivity and dynamic range.This approach is demonstrated in Example 4 (below), where the pH rangeover which the sensor operates and the size of the sensor response to agiven pH change are both controlled by altering the amount and nature ofthe ionising species incorporated into the polymer film. Although the insitu polymerisation method is preferable in many cases, other techniquesare appropriate for some materials and the scope of the invention is notlimited to this approach.

The following Examples further illustrate the invention.

Example 1

An Amylase Sensor

Starch gratings have the potential to be used as biosensors to detectthe concentration of the digestive enzyme alpha-amylase in a body fluid.This can be vitally important as an indication of acute pancreatitis.However starch does not lend itself to the production of ultra-finegrain photographic emulsion when using the conventional technique withgelatin, because it causes severe grain growth before the gelationoccurs.

Five steps are described for preparing and using the sensor. These are:

1. The treatment of glass to take a starch coating.

2. The preparation and coating of a layer of starch.

3. The introduction of a light-sensitive fine grain silver halidedeposit in the pre-coated starch layer by a diffusion process.

4. Exposure and development to record an optical hologram.

5. Use of the starch hologram as a sensor for amylase.

Step 1

Presubbing coating:

Solution A

50 ml deionised (DI) water 0.6 g potato starch hydrolysed forelectrophoresis (Sigma) 0.6 g Agarose Type A 0169 (Sigma)

The powdered carbohydrates are added to the cold water and stirred on aheater until the temperature reaches about 90° C. The mixture thenbecomes clear and free of any solid powder.

Solution B

0.10 g ammonium dichromate crystals 10 ml DI water

Solution B is added to Solution A to form a solution for a spin coating.

Clean microscope slide glass is then put in a standard laboratory spincoater and about 1 ml of the solution is pipetted onto the centre of theslide. The spin rate is adjusted to give a thickness of no more than 1μm. The lighting conditions during this part of the operation should beyellow or free of blue light.

The coating is thin, since a thicker layer may later cause a significantamount of holographic grating to be created in the sublayer. This wouldthen create spurious diffraction effects which might be confused withthe diffraction from the starch overlayer which should always besubstantially thicker than the sublayer.

The spin-coated material is dry within a minute or even a few seconds.Such spun coated slides are then exposed to a source of strongultraviolet light for a time sufficient to cause all the ammoniumdichromate in the film to crosslink the starch/agarose mix onto theglass slide. It is particularly important that the UV or violet lightfirst passes through the glass side rather than through the filmsurface.

Step 2

2.0 g potato starch hydrolysed for electrophoresis (Sigma) and 24 gwater are heated to 90° C. and stirred until clear. 1.0 ml 10% glutaricdialdehyde solution is added after the solution has cooled to about 50°C.

A relatively thick layer of coating is required on the subbed slides. Anumber of the subbed slides were lined up without gaps on a horizontalsurface and a warmed wire-wound Meyer bar is used (7 turns of wire percentimeter) to govern the coating thickness. The coating is then driedin a tepid air flow and the dry coating is heated at 116 C. for 1 hour,to obtain the necessary degree of crosslinking to enable a stableholographic grating to be formed after the treatment detailed below.

Step 3

Approx 1 ml of a 0.25M solution of silver nitrate is placed on a cleanflat surface and a starch-coated slide is pressed face down onto thedroplet. The solution then covers the starch coating by surface tension.The solution is left to soak into the starch layer for 2 minutes. It isthen removed, and surface liquid on the slide is removed by using filterpaper as blotting paper. The slide is then dried for a minute in astrong warm air flow.

36 g lithium bromide is stirred until dissolved in 900 ml water. This ispoured into 0.10 g sensitising dye that has been stirred into anddissolved in 300 ml methanol, in a 2 liter beaker and the solution isgiven a rapid rotation with a magnetic stirrer. The dried slide is heldin the rotated solution for a certain time. For convenience this timewill be called t(Br); depending on the conditions, its value may bechanged.

The starch coating is successfully impregnated with silver halide (inthis case AgBr) if t(Br) is 1 minute. If t(Br) is too long, then graingrowth can become a problem, but if t(Br) is too short, then thepenetration can be insufficient to obtain a later satisfactory gratin.After removing the slide from the bromide bath, it is at once washedunder running water.

If the dye is 1,1-diethyl-2,2′-cyanine iodide, then the slide will besensitized for exposure to a frequency-doubled YAG laser at 532 nmwavelength. If the dye is pinacyanol bromide, then the slide will besensitized to the 633 nm red from a HeNe laser. The dye does not have tobe in the solution of bromide ion at this point but may instead be usedin a separate bath after the precipitation of silver bromide has takenplace within the coated layer. However, by including the dye in thebromide ion bath, it causes a high light-sensitivity which has beenfound not to be so achievable if the dye bath is used separately.

The lithium bromide salt may be fully or partially replaced by equimolarequivalents of other halides (not fluorides) such as LiCl or LiI. Thiswill produce various alterations of the nature of the precipitatedsilver salt within the body of the coated layer. Also, in thisparticular example, all the lithium bromide may be replaced by sodiumbromide.

Step 4

The object used to make the hologram is a plain or curved mirror. Thestarch-coated plate, preferably in a swollen state, is placed close tothe mirror and a diverged laser beam is passed through the coating sothat it hits the mirror and is then reflected back through the coating,thus causing an interference pattern in the form of standing waves. Thisis recorded as a volume hologram with the interference fringes runningroughly parallel to the plane of the film (like pages in a book). Thisprinciple is well known to those familiar with holographic practices.

After exposure of the slide to the holographic recording conditions,preferably under water or other suitable liquid, the hologram isdeveloped in standard developers such as those detailed in PracticalHolography by Graham Saxby, published by Prentice Hall. The developmentcan be stopped by a 10% aqueous solution of acetic acid. Undevelopedsilver halide can then be removed in a 15% Hypo solution.

Step 5

The finished, processed and washed grating is cut to fit in aspectrophotometer cuvette. The liquid or sample to be tested for amylasecontent is added to the tube which should also contain appropriatebuffers to facilitate the enzyme reaction on the starch grating. Thespeed of attack at a given temperature is then monitored by recordingone of the characteristic changes to the reflection spectrum of thehologram as a function of time. This can be related to the concentrationof amylase in the original sample.

FIG. 1 shows the effect of α-amylase on the hologram. More specifically,the graph of reflectance (R) against wavelength (λ; nm) showsdegradation of the hologram. The traces are at 1 minute intervals, afterthe addition of 500 units amylase.

This approach may be extended to a wide range of other hydrolyticenzymes if the starch hologram is replaced by one made in a differentpolymer material which is cleavable by the enzyme of interest.

Example 2

An Ethanol Sensor

A microscope slide is presubbed as follows: a 1% solution of3-(trimethoxysilyl)propyl methacrylate in dry acetone is poured over itand left overnight to evaporate and hydrolyse on the glass surface.Excess silane is removed by washing with acetone before drying.

A solution of polymerizable monomers is prepared as follows:

475 μl 2-Hydroxyethyl methacrylate (HEMA) 25 μl Ethylene dimethacrylate(EDMA) 500 μl Propan-1-ol 5 mg 2,2′-dimethoxyphenyl acetophenone (DMPAP)

100 μl of this solution is poured onto a subbed slide (laidhorizontally) and covered with an inert sheet of non-stick as highdensity polythene of the type used for transfer lettering (Letraset).The sandwich is then exposed to UV light through the glass side untilfully polymerised. After removing the polythene cover sheet, the sampleis rinsed in methanol and dried in a warm air flow.

The sample is treated with silver nitrate as for Example 1, but thistime it is necessary for the 0.25 M silver nitrate to be in 50%water/50% 2-propanol to enable it to readily penetrate the polymer. Theslide is left in contact with the solution for an hour (even severalhours was not found to be detrimental or to make any difference).

After blotting the surface and drying as before, the slide is clampedand held in a rapidly rotating solution of bromide ions made up asfollows:

850 ml Methanol 0.03 g Sensitizing dye [see Example 1]

stirred until dissolved, then are added:

150 ml Water 27 g Lithium bromide

In this case immersion time t(Br) is 10 minutes. The slide is thenrinsed in running water.

After exposure, a developer formulation containing a large percentage ofalcohol is used:

25 g Sodium hydroxide 150 ml Water 10 g Hydroquinone 850 ml Methanol

The development is stopped in a bath of:

50 ml Acetic acid 150 ml Water 800 ml Methanol

The grating consists at this stage of developed silver fringes and itcan be advantageous to remove undeveloped silver bromide in a “fix”. Afix solution is 10% “hypo” (sodium thiosulphate) in a 50/50 solution ofmethanol/water. After agitating in hypo solution for 10 minutes,residual dye in the emulsion is also removed.

FIG. 2 is a graph of wavelength (λ; nm) against ethanol (V_(E1); vol %).It shows the change in volume of this hologram as it is immersed inmixtures of ethanol and water. It can therefore he used as a sensor tomonitor concentrations of ethanol.

Example 3

Na/K Sensors

Sensors capable of measuring the concentration of sodium ions in thepresence of potassium ions and vice versa are made.

3 mg DMPAP dissolved in 70 μl methanol [UV initiator] 50 μl HEMA 75 μlmethacryloyl ester of hydroxymethyl 12 crown 4

The liquid solution is poured over a presubbed microscope slide and thesame treatment is carried out as in Example 2, to produce a gratingwhich can be cut to suit a spectrometer cuvette or mounted at the end ofa fibre optic cable. Thus the silver grating is embedded in a copolymerof methacryloyl 12 crown 4 and HEMA in the approximate mole ratio of60:40. Since the grating is subjected to high concentrations of saltsolutions during preparation, it first requires extensive rinsing inseveral changes of de-ionized water for at least an hour before it canbe used as a sensor.

FIG. 3a is a graph of cation concentration ([+]; mM) against wavelengthshift (d_(λ); nm). It contrasts the effects of sodium ions () andpotassium ions (▴) on the response of the hologram.

By the same general procedure, but using the converse effect, a sensorcapable of measuring potassium ions in the presence of sodium ions canbe made by using a larger crown ether ring. Thus, if the 12 crown 4compound is substituted by an equivalent quantity of the equivalent 15crown 5 compound, the resulting grating can act as a potassium ionsensor. This is shown in FIG. 3b.

Example 4

A pH sensor

Sensors capable of measuring the pH of a liquid over a particular rangeare prepared in a similar manner to Example 2, but incorporatingfunctional monomers, which ionise over a particular pH interval. In thiscase, methacrylic acid (MAA) and vinyl pyridine (VP) are used. MAA is anacid and is uncharged at low pH. It ionises and becomes charged as thepH is raised. VP is charged at low pH and loses its charge as the pH israised.

The monomer compositions used to make the films are shown in thefollowing table:

HEMA EDMA MAA VP propan-1-ol DMPAP Identifier (μl) (μl) (μl) (μl) (μl)(mg) — 475 25 0 0 500 5  2% MAA 465 25 10 0 500 5  4% MAA 455 25 20 0500 5  6% MAA 445 25 30 0 500 5  8% MAA 435 25 40 0 500 5 10% MAA 425 2550 0 500 5  2% VP 465 25 0 10 500 5  4% VP 455 25 0 20 500 5  6% VP 44525 0 30 500 5  8% VP 435 25 0 40 500 5 10% VP 425 25 0 50 500 5

100 μl of each formulation is placed on a pre-subbed slide, covered witha polymer overlay and polymerised with U.V. light. The overlay isremoved, and each slide is washed with methanol. 100 μl 0.3M silverperchlorate in propan-1-ol:water (1:1) is placed on each slide, which iscovered with a polyester overlay to spread the liquid over the wholeslide area. This is left for 5 minutes, then the overlay is removed, thesurface blotted and the slide dried in warn air. Each slide is dipped ina bromide bath as described in Example 2. t(Br) was 2 minutes. Afterdipping, the slide is washed in running water.

During holographic exposure, it is important that the functional groupsare in their non-ionised form so that the subsequent replay wavelengthremains visible at all pH values. Hence MAA-containing films are exposedimmersed in 1% ascorbic acid, pH3, and VP-containing films in 0.1Mphosphate buffer, pH7.2. Development is with the methanolic hydroquinonedeveloper described in Example 2.

The holograms produced are tested in a series of citrate/phosphatebuffers with conductivity normalised to 20 mS/cm using KCl. Theresponses of the MAA-containing holograms are shown in FIG. 4a and thosefor VP-containing holograms in FIG. 4b. These graphs are each ofwavelength (λ; nm) against pH. It is clear that the pH interval overwhich the hologram responds can be changed by altering the nature of theionising functional group in the hologram. The size of the response, fora given change in pH, can be controlled by altering the amount ofionising monomer in the hologram.

Example 5

A Periodate Sensor

170 μl of 25% w/w methacrylamide in water and 284 μl of 50% w/wacrylamide in water are mixed. 11.56 mg methylene bisacrylamide wasadded and dissolved, followed by 50 μl of a 4% w/w solution in water ofa thioindigo vat dye in its leuco form. 100 μl of this mixture is placedon a pre-subbed slide and polymerised as for Example 2.

This is repeated twice, substituting for the methylene bisacrylamide for14.55 mg N,N ′-bisacryloylpiperazine and 15 mg1,2-dihydroxyethylenebisacrylamide respectively.

The films are treated to introduce silver nitrate as for Example 1 andthen dipped in a bromide bath as for Example 2. t(Br) is 1 minute. Thefilms are exposed, immersed in water and developed and fixed as forExample 1.

The resulting holograms are cut and placed in 1 ml water in cuvettes.The wavelength is monitored at 1 minute intervals for 10 minutes. Then10 μl 0.1M NaIO₄ is added and the response monitored for 30 minutes. Theresults are presented in FIG. 5, a graph of wavelength shift (d_(λ); nm)against time (t; min). It is clear that the methylenebisacrylamide ()and N,N′-diacryloylpiperazine (▴) cross-linked holograms are unaffectedby this treatment, but that the1,2-dihydroxyethylenebisacrylamide-containing hologram (▪) is caused toswell by the cleavage of the vicinal diol functionality in thecross-linker by the periodate anion. Hence the presence of periodate canbe measured by the hologram.

This Example can be extended by implication to any other chemically orbiochemically cleavable cross-linker built into an otherwise more orless inert polymer film.

What is claimed is:
 1. A method for the production of a holographicsensor wherein the holographic recording material forming the sensitiveelement is a polymer matrix, said method comprising providing thepolymer matrix; diffusing into the polymer matrix one or more solublesalts that undergo reaction in situ to form an insoluble photosensitiveprecipitate; and recording a holographic image.
 2. The method accordingto claim 1, wherein the polymer is a natural polymer.
 3. The methodaccording to claim 2, wherein the polymer is gelatin, starch or agarose.4. The method according to claim 1, wherein the polymer is a syntheticpolymer.
 5. The method according to claim 4, wherein the polymer isselected from the group consisting of polyvinyl alcohol;polyvinylpyrrolidone; polyhydroxyethyl acrylate;polyhydroxyethylmethacrylate; polyacrylamides; polymethacrylamides;homopolymers, or copolymers, or polymerisable derivatives of crownethers; and esters of, or co- or terpolymers of,polyhydroxyethylacrylate, polyhydroxyethylmethacrylate,polymethacrylamide and polyacrylamide; optionally with otherpolymerizable monomers or cross-linkers.
 6. The method according toclaim 1, wherein the polymer matrix is cross-linked.
 7. The methodaccording to claim 1, which additionally comprises, during a prior stepof forming the polymer, incorporating a component of the reaction in thepolymer.
 8. The method according to claim 1, which additionallycomprises incorporating in the sensor a photosensitizing dye, wherebythe spectral sensitivity is enhanced over a particular range ofwavelengths.
 9. The method according to claim 1, wherein thephotosensitive precipitate is a silver halide.
 10. The method accordingto claim 9, wherein the one or more soluble salts comprises a silversalt and the reaction comprises immersing the matrix in a solution of ahalide salt, optionally after drying the matrix.
 11. The methodaccording to claim 9, wherein the one or more soluble salts comprises ahalide salt, and the reaction comprises immersing the matrix in asolution of a silver salt, optionally after drying the matrix.
 12. Themethod according to claim 1, wherein the matrix interacts with itsphysical or chemical environment to create an optical response which isa change in the optical properties of the hologram.
 13. The methodaccording to claim 12, wherein the optical response arises from a volumechange in the matrix as a result of its interaction with an analyte orgroup of analytes.
 14. The method according to claim 12, wherein theoptical response arises from degradation or re-ordering of the matrix asa result of its interaction with an analyte or group of analytes. 15.The method according to claim 12, wherein the optical response arisesfrom a change in the distribution of refractive index within the matrixas a result of its interaction with an analyte or group of an analytes.16. The method according to claim 1, wherein the precipitate is ofparticles that have a grain size not more than 100 nm.
 17. The methodaccording to claim 16, wherein the grain size is less than 50 nm.
 18. Asensor for an analyte, the sensor comprising a hologram supported withina polymer matrix, wherein at least one optical characteristic of thehologram varies as a result of variation of a physical propertyoccurring throughout the bulk of the matrix, characterized in that thepolymer matrix is an insoluble polymer film.
 19. The sensor according toclaim 18, wherein the matrix is a synthetic polymer, e.g. a homopolymeror a copolymer of repeating monomer units.
 20. The sensor according toclaim 18, wherein the photosensitive substance is a salt.
 21. The sensoraccording to claim 18, wherein the polymer is a natural polymer.
 22. Amethod for the detection of an analyte in a sample, which comprisescontacting the sample with a sensor comprising a hologram supportedwithin a polymer matrix, wherein at least one optical characteristic ofthe hologram varies as a result of variation of a physical propertyoccurring throughout the bulk of the matrix, characterized in that thepolymer matrix is an insoluble polymer film, and observing an opticalcharacteristic of the hologram on the application of light.
 23. Themethod according to claim 22, wherein the photosensitive particles havea grain size not more than {fraction (1/10)} of the wavelength of theincident light.
 24. The sensor, according to claim 18, wherein thepolymer is gelatin, starch or agarose.
 25. The sensor, according toclaim 18, wherein the polymer is a synthetic polymer.
 26. The sensor,according to claim 25, wherein the polymer is selected from the groupconsisting of polyvinyl alcohol; polyvinylpyrrolidone; polyhydroxyethylacrylate; polyhydroxyethyl methacrylate; polyacrylamides;polymethacrylamides; homopolymers, or copolymers, of polymerisablederivatives of crown ethers; and esters of, or co- or terpolymers of,polyhydroxyethylacrylate, polyhydroxyethylmethacrylate,polymethacrylamide and polyacrylamide; optionally with otherpolymerizable monomers or cross-linkers.
 27. The Sensor, according toclaim 18, wherein the polymer matrix is cross-linked.
 28. The sensor,according to claim 18, further comprising a photosensitizing dye,whereby the spectral sensitivity is enhanced over a particular range ofwavelengths.
 29. The sensor, according to claim 18, wherein thephotosensitive precipitate is a silver halide.
 30. The sensor, accordingto claim 18, wherein the one or more soluble salts comprises a silversalt.
 31. The sensor, according to claim 18, wherein the one or moresoluble salts comprises a halide salt.
 32. The sensor, according toclaim 18, wherein the matrix interacts with its physical or chemicalenvironment to create an optical response which is a change in theoptical properties of the hologram.
 33. The sensor, according to claim32, wherein the optical response arises from a volume change in thematrix as a result of its interaction with an analyte or group ofanalytes.
 34. The sensor, according to claim 32, wherein the opticalresponse arises from degradation or re-ordering of the matrix as aresult of its interaction with an analyte or group of analytes.
 35. Thesensor, according to claim 32, wherein the optical response arises froma change in the distribution of refractive index within the matrix as aresult of its interaction with an analyte or group of analytes.
 36. Thesensor, according to claim 18, wherein the precipitate is of particlesthat have a grain size not more than 100 nm.
 37. The sensor, accordingto claim 36, wherein the grain size is less than 50 nm.